Assignment of functional amino acids around the active site of human DNA topoisomerase IIα

Y. Okada, Y. Ito, A. Kikuchi, Y. Nimura, S. Yoshida, Motoshi Suzuki

Research output: Contribution to journalArticle

17 Citations (Scopus)

Abstract

An expression library for active site mutants of human topoisomerase IIα (TOP2α) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2α mutants, no substitution was allowed at Tyr805, the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu794, Asp797, Ala801, and Arg804. Thus, these 5 residues are critical for human TOP2α activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser803 and Lys798, which confer drug resistance, lie adjacent to this plane.

Original languageEnglish
Pages (from-to)24630-24638
Number of pages9
JournalJournal of Biological Chemistry
Volume275
Issue number32
DOIs
Publication statusPublished - 11-08-2000

Fingerprint

Type II DNA Topoisomerase
Yeast
Catalytic Domain
Substitution reactions
Yeasts
Amino Acids
Temperature
Structural Models
DNA
Anchors
Catalysis
Drug Resistance
Human Activities
Oligonucleotides
Plasmids
Crystal structure
X-Rays
X rays
Substrates
Pharmaceutical Preparations

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Okada, Y. ; Ito, Y. ; Kikuchi, A. ; Nimura, Y. ; Yoshida, S. ; Suzuki, Motoshi. / Assignment of functional amino acids around the active site of human DNA topoisomerase IIα. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 32. pp. 24630-24638.
@article{20a3bf752b5f41cba7dedd38dabf878d,
title = "Assignment of functional amino acids around the active site of human DNA topoisomerase IIα",
abstract = "An expression library for active site mutants of human topoisomerase IIα (TOP2α) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2α mutants, no substitution was allowed at Tyr805, the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu794, Asp797, Ala801, and Arg804. Thus, these 5 residues are critical for human TOP2α activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser803 and Lys798, which confer drug resistance, lie adjacent to this plane.",
author = "Y. Okada and Y. Ito and A. Kikuchi and Y. Nimura and S. Yoshida and Motoshi Suzuki",
year = "2000",
month = "8",
day = "11",
doi = "10.1074/jbc.M003243200",
language = "English",
volume = "275",
pages = "24630--24638",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "32",

}

Assignment of functional amino acids around the active site of human DNA topoisomerase IIα. / Okada, Y.; Ito, Y.; Kikuchi, A.; Nimura, Y.; Yoshida, S.; Suzuki, Motoshi.

In: Journal of Biological Chemistry, Vol. 275, No. 32, 11.08.2000, p. 24630-24638.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Assignment of functional amino acids around the active site of human DNA topoisomerase IIα

AU - Okada, Y.

AU - Ito, Y.

AU - Kikuchi, A.

AU - Nimura, Y.

AU - Yoshida, S.

AU - Suzuki, Motoshi

PY - 2000/8/11

Y1 - 2000/8/11

N2 - An expression library for active site mutants of human topoisomerase IIα (TOP2α) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2α mutants, no substitution was allowed at Tyr805, the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu794, Asp797, Ala801, and Arg804. Thus, these 5 residues are critical for human TOP2α activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser803 and Lys798, which confer drug resistance, lie adjacent to this plane.

AB - An expression library for active site mutants of human topoisomerase IIα (TOP2α) was constructed by replacing the sequence encoding residues 793-808 with a randomized oligonucleotide cassette. This plasmid library was transformed into a temperature-sensitive yeast strain (top2-1), and viable transformants were selected at the restrictive temperature. Among the active TOP2α mutants, no substitution was allowed at Tyr805, the 5' anchor of the cleaved DNA, and only conservative substitutions were allowed at Leu794, Asp797, Ala801, and Arg804. Thus, these 5 residues are critical for human TOP2α activity, and the remaining mutagenized residues are less critical for function. Using the x-ray crystal structure of yeast TOP2 as a structural model, it can be deduced that these 5 functionally important residues lie in a plane. One of the possible functions of this plane may be that it interacts with the DNA substrate upon catalysis. The side chains of Ser803 and Lys798, which confer drug resistance, lie adjacent to this plane.

UR - http://www.scopus.com/inward/record.url?scp=0034637574&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034637574&partnerID=8YFLogxK

U2 - 10.1074/jbc.M003243200

DO - 10.1074/jbc.M003243200

M3 - Article

VL - 275

SP - 24630

EP - 24638

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 32

ER -